Cheluminate-HRP PicoDetect

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1 Cheluminate-HRP PicoDetect Enhanced ChemiLuminescence Detection Kit for Western / Southern / Northern Blotting Product-No. A3417 Description The CheLuminate-HRP PicoDetect is a complete kit with ready-to-use reagents for chemiluminescent detection of immobilized proteins (Western blot) or immobilized nucleic acids (Southern or Northern blot), conjugated with horseradish peroxidase (HRP) directly or indirectly. The use of enhanced chemiluminescence was introduced by Thorpe and Kricka (1, 2). In the presence of hydrogen peroxide (H 2 O 2 ), HRP catalyzes the oxidation of cyclic diacylhydrazides, such as luminol. Immediately following the oxidation, the luminol is in an excited state (intermediate reaction product), which decays to the ground state by emitting light. Strong enhancement of the light emission is produced by enhancers, such as phenolic compounds. This kit consists of two solutions: Solution A (contains luminol and enhancer) and solution B (stable H 2 O 2 solution). For the detection assay, both solutions are mixed in a 1 : 1 ratio and 100 µl/cm² are applied. Advantages of using this kit: The CheLuminate-HRP PicoDetect kit corresponds to the classic chemiluminescence substrates based on phenolic enhancers and is comparable to the ECL System in terms of the chemistry and the substrate of choice for detection of highly expressed proteins in combination with economical (standard) antibodies. CheLuminate-HRP PicoDetect bears a low risk of signal-saturation which simplifies signal detection via autoradiography film. 1-2 hours of light emission allow sufficient time to optimize exposure conditions. MOST ECONOMIC much less costly than other chemiluminescent substrates SUPERIOR FOR FILM for standard Western blot detection on autoradiography film EASY TO HANDLE picogram limit of detection allows detection of highly expressed proteins without background problems HIGH STABILITY working solution is stable for 7 days at RT Kit Reagents: Keep always protected from light! Ordering information Cheluminate-HRP PicoDetect Prod. No. Size (sufficient for) Solution A (luminol/enhancer) Solution B (peroxide solution) A3417, cm 2 A3417,1200A 60 ml A3417,1200B 60 ml A3417, cm 2 A3417,5000A 250 ml A3417,5000B 250 ml Storage: 2-8 C. We recommend mixing the required volume of solutions A + B just before use. The mixture is stable for up to 7 days. It is important to bring the mix to room temperature before use! In case you don't get a signal with this premixed solution, add 25 µl per 5 ml mix of a 3 % solution of hydrogen peroxide. Caution: If above solutions come into contact with eyes or skin, flush with plenty of water and remove contaminated clothing. Quality Control For the quality control of the EnhancedChemiluminescence kit the following is used: Antigen: Bovine serum albumin (BSA) Primary antibody: Rabbit anti BSA Secondary antibody: Goat anti rabbit IgG- HRP The detection limit is in the picogram range. References: (1) Thorpe, G.H.G. & Kricka, L.J. (1986) Methods Enzymol. 133, (2) Thorpe, G.H.G. et al. (1985) Clin. Chem. 31,

2 JBV Principles of protein detection procedure: Antigen Membrane 1. Antibody 2. Antibody HRP Luminol/H 2 O 2 Enhancer Light Film/ Imaging System Protocol for Western Blotting and Chemiluminescence Detection 1. Preparation of Solutions (not supplied with the kit) 1.1 Tris Buffered Saline (TBS) 6.05 g Tris base (50 mm; Product-No.: A1086) 8.76 g sodium chloride (150 mm; Product No.: A1149) Adjust ph to 7.5 with hydrochloric acid (Product-No.: A0659) Add distilled water up to 1000 ml 1.2 Phosphate Buffered Saline (PBS; Product-No.: A0964) - optional 11.5 g di-sodium hydrogen phosphate, anhydrous (80 mm) 2.96 g sodium dihydrogen phosphate (20 mm) 5.84 g sodium chloride (100 mm) Add distilled water up to 1000 ml Check ph (should be 7.5) 1.3 TBS-Tween (TBS-T) and PBS-Tween (PBS-T) Dilute 1 ml of Tween 20 (Product-No.: A1389) in 1000 ml of buffer (0.1 % final concentration). 1.4 A sufficient volume of wash buffer, blocking buffer and antibody solution should be used to cover the blot to ensure that the membrane does not become dry. This will also ensure a reduced non-specific background. 1.5 Do not use sodium azide as a preservative for the secondary antibody dilutions, as azide irreversibly inhibits horseradish peroxidase. 1.6 Wherever use of dried milk is indicated, this can be substituted with low-fat milk. 2. Electrophoresis, Blotting and Membrane Preparation 2.1 Carry out electrophoresis for protein separation. Either non-denaturing gel, SDS-PAGE or two dimensional gels may be used Transfer proteins from the gel to a membrane. Use nitrocellulose or PVDF membrane. PVDF membranes must be wetted briefly in methanol then soaked in distilled water for 1-3 minutes, followed by equilibration in transfer buffer. 2.3 Membrane Blocking Block non-specific binding sites by incubating the membrane for 1 hour at room temperature with shaking in TBS-T or PBS-T solution containing 5 % dried milk (w/v; Product-No.: A0830). This step can be performed overnight at +4 C without shaking. 2.4 Primary Antibody Dilute the primary antibody in TBS-T or PBS-T with 2 % dried milk (w/v). Incubate the membrane in the solution for 1 hour at room temperature with shaking, or overnight at +4 C without shaking. 2.5 Membrane Washing Wash the membrane three times in TBS-T or PBS-T for 10 minutes each. Use at least 50 ml of buffer for 10 x 10 cm membrane. 2.6 Secondary Antibody Dilute the HRP-labeled secondary antibody in TBS-T or PBS-T with 2 % dried milk (w/v). Incubate the membrane in the solution for 1 hour at room temperature with shaking. 2.7 Membrane Washing Wash the membrane as detailed in

3 3. Enhanced Chemiluminescence Detection 3.1 Preparations Prepare the following equipment and solutions in a dark room: - X-ray film cassette - X-ray film - Timer - Developer, fixer and water in tanks - Transparent plastic bag or saran wrap - Glass pipettes - Sterile gloves - to prevent hand contact with membrane, film or reagents 3.2 Detection Mix an equal volume of Solution A and Solution B to give sufficient solution to cover the membrane (0.1 ml/cm 2 ). Let the detection mix equilibrate for at least 5 minutes Drain the excess buffer from the washed blots. Do not let the membrane dry out. Add the detection mix directly to the blot (protein side up). Incubate for 1-3 minutes at room temperature Drain off excess detection mix and wrap the membrane in saran wrap. Gently remove air pockets Place the blots, protein side up, in the film cassette. Switch off the lights and use red safety light. Place a sheet of film on the blot, close the cassette and expose for seconds Replace the exposed film with a new one, close the cassette and develop the first exposed film Expose the second film for a suitable time according to the signal intensity on the first film If signal intensity was too high, wait up to 30 minutes before re-exposing. 4. Optimization of Antibody Concentration for Enhanced Chemiluminscence Detection It is essential to optimize the immunoblot conditions to achieve maximum signal and minimum background. First optimize the concentration of the primary antibody using a constant amount of secondary-hrp conjugate. Using the optimized primary antibody concentration, adjust the concentration of the secondary anti-body-hrp conjugate. 4.1 Dot-Blot for Primary Antibody Optimization Prepare one piece of nitrocellulose membrane for each primary antibody dilution to be tested Spot a dilution range of protein onto the membrane Allow the membrane to air-dry Block non-specific binding sites by incubating the strip for 1 hour at room temperature with shaking in TBS-T or PBS-T solution containing 5 % dried milk (w/v). This step can be performed overnight at +4 C without shaking Prepare several dilutions of primary antibody in TBS-T or PBS-T with 2 % dried milk (w/v), (e.g. 1:100 1:5000). Incubate one piece of membrane in each dilution for 1 hour at room temperature with constant shaking, or overnight at +4 C without shaking Wash the membranes three times in TBS-T or PBS-T for 10 minutes each. Use at least 0.5 ml of buffer per 1 cm 2 membrane Dilute the HRP-labeled secondary antibody in TBS-T or PBS-T with 2 % dried milk (w/v) to the known optimal dilution. Incubate each strip in the solution for 1 hour at room temperature with shaking Wash the membranes as detailed in above Detection: as detailed in 3.2 above. 4.2 Dot-Blot for Secondary Antibody Optimization Prepare one piece of nitrocellulose membrane for each secondary antibody dilution to be tested Prepare dot-blots as detailed in above Dilute the primary antibody in TBS-T or PBS-T with 2 % dried milk (w/v) to the known optimal dilution. Incubate each strip in the solution for 1 hour at room temperature with shaking Wash the membranes as detailed in above Prepare several dilutions of secondary antibody in TBS-T or PBS-T with 2 % dried milk (w/v), e.g. 1:5,000 1:100,000). Incubate one piece of membrane in each dilution for 1 hour at room temperature with constant shaking Wash the membranes as detailed in above Detection: as detailed in 3.2 above. 3

4 5. Stripping and Reprobing of Membrane The immunoblot can be stripped of blocking reagent and antibodies, and then reprobed as required. 5.1 Incubate membrane in stripping buffer for 30 minutes at C. Stripping buffer: 62.5 mm Tris-HCl ph 6.8 (Product-No.: A1087) 100 mm -mercaptoethanol (Product-No.: A1108) 2 % (w/v) SDS (Product-No.: A1112) 5.2 Wash the membrane twice in TBS-T or PBS-T for 10 minutes each. Use at least 50 ml of buffer for 10 x 10 cm membrane. To ensure removal of antibodies, incubate the membrane with the detection reagents and expose against film. Repeat previous steps if a signal is detected. 5.3 Reprobe the blot as detailed in above. Short Protocol for Proteins Step Action Volume Time Remarks Electrophoresis and Blotting According to usual protocols use Nitrocellulose or PVDF membrane Membrane Blotting Block membrane with blocking solution, TBS-T or PBS-T with 5 % dried milk (w/v), under constant shaking hour at room temperature Alternatively: overnight at +4 C without shaking Primary Antibody Dilute the primary antibody in TBS-T or PBS- T with 2 % dried milk (w/v). Incubate the membrane in solution with shaking hour at room temperature Alternatively: overnight at +4 C without shaking Washing Three times with TBS-T under constant shaking x 10 minutes Secondary Antibody Dilute the HRP-labeled secondary antibody (1 : : 60000) in TBS-T or PBS-T with 2 % dried milk (w/v). Incubate the membrane in the solution hour at room temperature Washing Three times with TBS-T under constant shaking x 10 minutes Equilibration Mix equal volumes of Solution A and B (A3417) minutes Detection Incubate membrane in detection mix solution minutes With gentle shaking Exposure Remove excess detection mix, wrap in saran wrap and expose to film minutes Remove air pockets Your Blots DON T HAVE to look like these! Please check our Optimization and Troubleshooting Guide for Chemiluminescence Detection in Western Blotting: 4

5 Principles of nucleic acid detection procedure: A U Biotin G C C G T A Membrane Probe Streptavidin HRP Luminol/H 2 O 2 Enhancer Light Film/ Imaging System Protocol for Southern/Northern Blotting and Chemiluminescence Detection 1. Preparation of Solutions (not supplied with the kit) 1.1 Tris-buffered saline ph 7.5 (Buffer A) g Tris base (100 mm; Product-No.: A2264) g sodium chloride (600 mm; Product No.: A2942) Adjust ph to ph 7.5 with hydrochloric acid (Product-No.: A0659). Add DEPC-treated water up to 1000 ml. 1.2 Wash Buffer No. 1 2X SSC (Product-No.: A1396) 0.1 % SDS (Product-No.: A1112) 1.3 Wash Buffer No X SSC 0.1 % SDS % Blocking Reagent in Buffer A 0.2 g Blocking Reagent CA (Order-No. A3409,0010) 100 ml Buffer A ph 7.5 (1.1) Heat in water bath or microwave to C. Mix well % Tween 20 in Buffer A 0.5 ml Tween 20 (Product-No.: A1389) 500 ml Buffer A ph 7.5 Mix well % Blocking Reagent CA in Buffer A 0.5 g Blocking Reagent CA (Order-No. A3409,0010) 100 ml Buffer A ph 7.5 Heat in a water bath or microwave to C. Mix well. Notes: Do not use sodium azide as a preservative for the streptavidin-hrp dilution, since azide irreversibly inhibits Horseradish peroxidase. Non-fat dry milk inhibits the streptavidin-biotin interaction, due to its content of biotin. Blocking Reagent CA is free of biotin! Probe concentration, which is too high, will often lead to background. Therefore, the probe concentration should not be increased above the recommended concentrations. (The recommended final probe concentration is 2 10 ng/ml or 1 2 x 10 6 cpm/ml for Northern or Southern hybridization). 5

6 2. Electrophoresis Blotting and Membrane Preparation 2.1 Carry out electrophoresis for nucleic acid separation. 2.2 Denature the DNA by soaking the gel for 45 minutes in several volumes of 1.5 M NaCl; 0.5 N NaOH with constant, gentle agitation. 2.3 Rinse the gel briefly in deionized water, and neutralize it by soaking for 30 minutes in several volumes of a solution of 1 M Tris ph 7.4, 1.5 M NaCl at room temperature with constant, gentle agitation. Change the neutralization solution and continue soaking the gel for a further 15 minutes. Notes: Nylon membrane binds small DNA fragments more efficiently than nitrocellulose membranes. Fragments of less than 300 nucleotides in length are not retained by 0.45 µm nitrocellulose membranes. (Use a pore size of 0.2 µm). Use gloves and blunt-ended forceps to handle the membrane. 2.4 Soak the nitrocellulose membrane in deionized water until completely wet. Immerse the membrane in transfer buffer (20X SSC or 20X SSPE; Product-No.: A1397). 2.5 Transfer the nucleic acids from the gel to a membrane for 2-24 hours. Mark the positions of the gel slots on the filter with a very soft lead pencil or a ball point pen. 2.6 After transfer soak the membrane in 6X SSC for 5 minutes at room temperature (this removes any pieces of agarose sticking to the membrane). 2.7 Remove the membrane from the 6X SSC and allow excess fluid to drain away. Place the membrane flat on a paper towel to dry for at least 30 minutes at room temperature. 2.8 Sandwich the filter between two sheets of dry 3MM paper. Fix the DNA to the filter by baking for 30 minutes to 2 hours at 80 C in a vacuum oven. 2.9 Hybridization using the Hybridization Solution with non-radioactively labeled probes Warm the Hybridization Solution (Order-No.: A3728,0100) at 68 C for Northern and at 60 C for Southern, and stir well to completely dissolve any precipitate Prehybridize membranes in a minimum of 0.1 ml/cm 2 of Hybridization Solution with continuous shaking at 68 C for Northern and at 60 C for Southern for minutes. The volume of solution must be sufficient to completely cover the membrane, or high backgrounds may result Denature the non-radioactively labeled DNA probe at C for 2-5 minutes. Chill quickly on ice Add non-radiolabeled probe to a sufficient volume of fresh Hybridization Solution. Mix gently. For recommended final probe concentrations, see notes above Replace the Hybridization Solution with the fresh solution containing the non-radiolabeled DNA probe. Remove all air bubbles from the container, and make sure the Hybridization Solution is evenly distributed over the entire blot Hybridize with continuous shaking at 68 C for Northern and at 60 C for Southern for hours. (For high target applications, shorter hybridization times can be used. For single-gene sequences, hybridization can be performed overnight) Wash the membranes at room temperature twice, 15 minutes each time, with at least 0.5 ml/cm 2 of 2X SSC, 0.1 % SDS (Wash No. 1) Wash the membrane twice at 68 C for Northern and at 60 C for Southern, 15 minutes each time, with at least 0.5 ml/cm 2 of 1-0.1X SSC, 0.1 % SDS, with continuous agitation. Note: These washing conditions may be too stringent for probes that are not completely homologous to the target. If this is the case, lower the temperature to 50 C Remove the blot with forceps and shake off excess wash solution. Rinse the blot in a large amount (2 ml/cm 2 ) of Buffer A ph Incubate the blot in 0.2 % Blocking Reagent CA in Buffer A for 30 minutes at room temperature with gentle agitation Incubate the blot in diluted streptavidin-hrp (1 : : 3000) in 0.5 % Blocking Reagent CA in Buffer A for 30 minutes at room temperature with gentle agitation (minimum ml/cm 2 ) Wash the blot three times in 0.1 % Tween 20 in Buffer A, for 10 minutes each time. Use at least 2 ml/cm 2 of buffer. 6

7 3. Enhanced Chemiluminescence Detection 3.1 Preparations Prepare the following equipment and solutions in a dark room: - X-ray film cassette and X-ray film - Timer, pipette and tips, transparent plastic bag or saran wrap - Developer, fixer and water in tanks - Sterile gloves - to prevent hand contact with membrane, film or reagents 3.2 Detection Mix an equal volume of Solution A and Solution B to give sufficient solution to cover the membrane (0.1 ml/cm 2 ). Let the detection mix equilibrate for at least 5 minutes Drain the excess buffer from the washed blots. Do not let the membrane dry out. Add the detection mix directly to the blot (nucleic acid side up). Incubate for 1-3 minutes at room temperature Drain off excess detection mix and wrap the membrane in saran wrap. Gently remove air pockets Place the blots (nucleic acid side up) in the film cassette. Switch off the lights and use red safety light. Place a sheet of film on the blot, close the cassette and expose for seconds Replace the exposed film with a new one, close the cassette and develop the first exposed film Expose the second film for a suitable time according to the signal intensity on the first film If signal intensity was too high, wait up to 30 minutes before re-exposing. Short Protocol for Nucleic acids Step Action Volume Time Remarks Electrophoresis Blotting According to usual protocols Immerse the membrane in ddh 2O and then transfer buffer Transfer according to the usual protocol use Nitrocellulose or nylon membrane Membrane Washing and Fixation Pre-hybridization and Hybridization Washing (I) Washing (II) Blocking - soak the membrane in 6xSSC - dry for at least 30 minutes - fixation by baking or by U.V. crosslinking According to the manufacturer's instructions. Use denatured biotin-labeled DNA probe Twice with 2xSSC, 0.1 % SDS (Wash No. 1) Twice with 1-0.1xSSC, 0.1 % SDS (Wash No. 2) Immerse the membrane in Buffer A Incubate the blot in 0.2 % Blocking Reagent CA (A3409) in Buffer A min. at RT - RT hrs at 80 C or 32 sec erg hrs at 68 C or 60 C x 15 min. at RT x 15 min. at 68 C or 60 C min. at RT Streptavidin-HRP Dilute the streptavidin-hrp (1 : : 10000) in 0.5 % Blocking Reagent CA in Buffer A. Incubate the membrane in the solution minutes at RT Washing Equilibration Detection Exposure Three times with 0.1 % Tween 20 in Buffer A Mix equal volumes of Solution A and B (A3417) Incubate membrane in detection mix solution Remove excess detection mix, wrap in saran wrap and expose to film 2 3 x 10 minutes minutes minutes With gentle shaking minutes Remove air pockets erg = The unit of energy in the centimeter-gram-second system. The work performed by a force of 1 dyne acting through a distance of 1 centimeter. This energy unit is used with the UV cross-linkers. 7

8 CheLuminate-HRP Substrates & Related products Selection Guide: CheLuminate-HRP Substrates for HRP detection in Immunoblots highly expressed proteins medium expressed proteins poorly expressed proteins Classical ECL technology New Generation Kits, employing an additional secondary enhancer Product A3417 A7786 A7807 A7879 CheLuminate-HRP PicoDetect CheLuminate-HRP PicoDetect Extended CheLuminate-HRP FemtoDetect CheLuminate-HRP FemtoDetect Plus Detection Limit Picogram Low-picogram (10-12 ) High-femtogram (10-13 ) Low-femtogram (10-15 ) Highlights CLASSIC (light output based on phenolic enhancer) Easy-to-handle ECONOMICAL LONG AND STEADY LIGHT EMISSION ECONOMICAL EXTENDED SIGNAL DURATION (multiple exposure for high quality blots) Signal Duration 1-2 hours 6 hours 12 hours 8 hours MOST SENSITIVE (at least 150 times more intense than standard substrates) EXTENDED SIGNAL DURATION Suggested antibody dilution (based on 1 mg/ml stock solution) Primary: 1:100-1:5,000 Secondary: 1:5,000-1:100,000 Primary: 1:500-1:5,000 Secondary: 1:20,000-1:100,000 Primary: 1:1,000-1:15,000 Secondary: 1:25,000-1:150,000 Primary: 1:5,000-1:100,000 Secondary: 1:100,000-1:500,000 Excellent alternative to ECL SuperSignal WestPico WesternLightning PLUS Luminata Classic Lumi-Light LumiGLO LiteAblot PLUS SERVALightPolaris PicoMax ; PicoTect Immun-Star HRP ECL SuperSignal WestPico WesternLightning PLUS Luminata Classico Lumi-Light LumiGLO LiteAblot PLUS SERVALightPolaris PicoMax ; PicoTect Immun-Star HRP ECL Prime SuperSignal WestDura WesternLightning PRO Luminata Crescendo Lumi-Light PLUS LiteAblot EXTEND SERVALightEos PCD ECL WesternBright Immun-Star WesternC ECL Select SuperSignal WestFemto WesternLightning Ultra Luminata Forte LumiGLO Reserve LiteAblot TURBO SERVALightHelios FemtoMax Related Products A5239 Pure Nitrocellulose unsupported 0.45 μm Transfer Membrane A5243 PVDF-Star Transfer Membrane 0.45 μm A1391 Albumin Fraction V (ph 7.0) A0830 Nonfat dried milk powder A1389 Tween 20 BioChemica A5001 TBS (Tris-buffered saline) (20X) - Powder A9201 PBS tablets ph 7.4 (for 1 L) A7099 Blocking Buffer I A7516 Blocking Buffer II EGrade A7252 Blocking Buffer III BSA A6485 CrossDown Buffer 8

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